The present application claims priority under 35 U.S.C. xc2xa7119 to German Application 199 51 708.8, filed on Oct. 27, 1999.
The present invention provides nucleotide sequences coding for the export of branched-chain amino acids, a process for the identification and isolation thereof and a process for the fermentative production of branched-chain amino acids using coryneform bacteria in which genes which code for the export of branched-chain amino acids are amplified.
The branched-chain amino acids L-isoleucine, L-valine and L-leucine are used in the pharmaceuticals industry, in human medicine and in animal nutrition.
It is known that branched-chain amino acids may be produced by fermentation of strains of coryneform bacteria, in particular Corynebacterium glutamicum. Due to their great significance, efforts are constantly being made to improve the production process. Improvements to the process may relate to measures concerning fermentation technology, for example stirring and oxygen supply, or to the composition of the nutrient media, such as for example sugar concentration during fermentation, or to working up of the product by, for example, ion exchange chromatography, or to the intrinsic performance characteristics of the microorganism itself.
The performance characteristics of these microorganisms are improved using methods of mutagenesis, selection and mutant selection. In this manner, strains are obtained which are resistant to antimetabolites, such as for example the isoleucine analogue isoleucine hydroxyamate (Kisumi M, Komatsubara S, Sugiura, M, Chibata I (1972) Journal of Bacteriology 110: 761-763), the valine analogue 2-thiazolealanine (Tsuchida T, Yoshinanga F, Kubota K, Momose H (1975) Agricultural and Biological Chemistry; Japan 39: 1319-1322) or the leucine analogue xcex1-aminobutyrates (Ambe-Ono Y, Sato K, Totsuka K, Yoshihara Y, Nakamori S (1996) Bioscience Biotechnology Biochemistry 60: 1386-1387) or which are auxotrophic for regulatorily significant metabolites and produce branched-chain amino acids (Tsuchida T, Yoshinaga F, Kubota K, Momose H, Okumura S (1975) Agricultural and Biological Chemistry; Nakayama K, Kitada S, Kinoshita S (1961) Journal of General and Applied Microbiology, Japan 7: 52-69; Nakayama K, Kitada S, Sato Z, Kinoshita (191) Journal of General and Applied Microbiology, Japan 7: 41-51).
For some years, the methods of recombinant DNA technology have also been used for improvement of strains of Corynebacterium which produce branched-chain amino acids by amplifying individual biosynthesis genes for branched-chain amino acids and investigating the effect on branched-chain amino acid production. Review articles on this subject may be found inter alia in Kinoshita (xe2x80x9cGlutamic Acid Bacteriaxe2x80x9d, in: Biology of Industrial Microorganisms, Demain and Solomon (Eds.), Benjamin Cummings, London, UK, 1985, 115-142), Hilliger (BioTec 2, 40-44 (1991)), Eggeling (Amino Acids 6:261-272 (1994)), Jetten and Sinskey (Critical Reviews in Biotechnology 15, 73-103 (1995)), Sahm et al. (Annuals of the New York Academy of Science 782, 25-39 (1996)), and Eggeling et al., Journal of Biotechnology 56: 168-180 (1997)).
The inventors set themselves the object of providing novel measures for the improved fermentative production of branched-chain amino acids.
Branched-chain amino acids are used in the pharmaceuticals industry, in human medicine and in animal nutrition. There is accordingly general interest in providing novel improved processes for the production of branched-chain amino acids.
Any subsequent mention of branched-chain amino acids should be taken to mean in particular L-isoleucine, L-valine or L-leucine.
The present invention provides isolated polynucleotides containing at least one polynucleotide sequence selected from the group
a) polynucleotide which is at least 70% identical to a polynucleotide which codes for a polypeptide containing at least one amino acid sequence SEQ ID no. 3 or 5,
b) polynucleotide which codes for a polypeptide which contains an amino acid sequence which is at least 70% identical to the amino acid sequence of SEQ ID no. 3 or 5,
c) polynucleotide which is complementary to the polynucleotides of a) or b) and
d) polynucleotide containing at least 15 successive bases of the polynucleotide sequences of a), b) or c).
The present invention also provides preferably recombinant DNA replicable in coryneform bacteria and originating from Corynebacterium which contains at least the nucleotide sequences which code for the genes brnF and/or brnE, as shown in SEQ ID no. 1 and in SEQ ID no. 6.
The present invention also provides replicable DNA as described in a)-c) above containing:
(i) the nucleotide sequences shown in SEQ ID no. 1 or SEQ ID no. 6 which code for the genes brnE and/or brnF, or
(ii) at least one sequence which matches the sequence (i) within the degeneration range of the genetic code, or
(iii) at least one sequence which hybridises with the complementary sequence to sequences (i) or (ii) and optionally
(iv) functionally neutral sense mutations in (i).
The present invention also provides
polynucleotides as in a)-d) above which comprise recombinant DNA replicable in coryneform bacteria and contain containing at least one of the nucleotide sequences selected from those shown in SEQ ID no. 1, 2, 4 or 6
polypeptides as described in a)-d) above which comprimise recombinant DNA replicable in coryneform bacteria which code for polypeptides which contain at least one of the amino acid sequences as shown in SEQ ID no. 3 or 5
a vector containing the polynucleotide or polynucleotides as described in a)-c) above or the DNA sequence shown in SEQ ID no. 1 or SEQ ID no. 6.
and coryneform bacteria acting as host cell which contain the vector.
The present invention also provides polynucleotides which substantially consist of one polynucleotide sequence, which are obtainable by screening by means of hybridisation of a suitable gene library, which contains the complete genes having the polynucleotide sequences according to SEQ ID no. 1, 2, 4 or 6 with a probe which contains the sequence of the stated polynucleotides according to SEQ ID no. 1, 2, 4 or 6 or a fragment thereof and isolation of the stated DNA sequences.
Polynucleotide sequences according to the invention are suitable as hybridisation probes for RNA, cDNA and DNA in order to isolate full length cDNA which codes for isoleucine, leucine or valine export proteins and to isolate such cDNA or genes, the sequences of which exhibit a high level of similarity with that of the brnF and/or brnE gene.
Polynucleotide sequences according to the invention are furthermore suitable as primers, with the assistance of which, usingthe polymerase chain reaction (PCR), DNA of genes whichcode for isoleucine, leucine or valine export proteins may be produced.
Such oligonucleotides acting as probes or primers contain at least 30, preferably at least 20, very particularly preferably at least 15 successive nucleotides. Oligonucleotides having a length of at least 40 or 50 base pairs are also suitable.
xe2x80x9cIsolatedxe2x80x9d means separated from its natural environment.
xe2x80x9cPolynucleotidexe2x80x9d generally relates to polyribonucleotides and polydeoxyribonucleotides, wherein the RNA or DNA may be unmodified or modified.
xe2x80x9cPolypeptidesxe2x80x9d are taken to mean peptides or proteins which contain two or more aminoacids connected by peptide bonds.
The polypeptides according to the invention include the polypeptides according to SEQ ID no. 3 and/or 5, in particular those having the biological activity of transporting branched-chain amino acids and also those which are at least 70% identical to the polypeptides according to SEQ ID no. 3 and/or 5, preferably at least 80% and in particular 90% to 95% identical to the polypeptides according to SEQ ID no. 3 and/or 5 and exhibit the stated activity.
The present invention also provides coryneform microorganisms, in particular of the genus Corynebacterium, transformed by the introduction of the stated replicable DNA.
The invention furthermore relates to a process for the fermentative production of branched-chain amino acids using coryneform bacteria, which in particular already produce the branched-chain amino acids and in which the nucleotide sequences of the genes brnE and/or brnF which code for the export of branched-chain amino acids are amplified, in particular overexpressed.
In this connection, the term xe2x80x9camplificationxe2x80x9d describes the increase in the intracellular activity of one or more enzymes (proteins) in a microorganism, which enzymes are coded by the corresponding DNA, for example by increasing the copy number of the gene or genes, by using a strong promoter or a gene which codes for a corresponding enzyme (protein) having elevated activity and optionally by combining these measures.
The microorganisms, provided by the present inventions, may produce branched-chain amino acids from glucose, sucrose, lactose, mannose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. The microorganisms may comprise representatives of the coryneform bacteria in particular of the genus Corynebacterium. Within the genus Corynebacterium, Corynebacterium glutamicum may in particular be mentioned, which is known in specialist circles for its ability to produce L-amino acids.
Suitable strains of the genus Corynebacterium, in particular of the species Corynebacterium glutamicum, are in particular the known wild type strains
Corynebacterium glutamicum ATCC13032
Brevibacterium flavum ATCC14067
Brevibacterium lactofermentum ATCC13869 and
Brevibacterium divaricatum ATCC14020
and branched-chain amino acid producing mutants or strains produced therefrom,
such as for example the isoleucine producing strains
Corynebacterium glutamicum ATCC14309
Corynebacterium glutamicum ATCC14310
Corynebacterium glutamicum ATCC14311
Corynebacterium glutamicum ATCC15168
Corynebacterium ammoniagenes ATCC 6871,
such as for example the leucine producing strains
Corynebacterium glutamicum ATCC 21885
Brevibacterium flavum ATCC 21889
or such as for example the valine producing strains
Corynebaccterium glutamicum DSM 12455
Corynebarcterium glutamicum FERM-P 9325
Brevibacterium lactofermentum FERM-P 9324
Brevibacterium lactofermentum FERM-BP 1763.
The inventors succeeded in isolating the novel genes brnE and brnF from Corynebacterium glutamicum. The genes are isolated by initially producing a mutant of C. glutamicum which is defective with regard to brnF or brnE. To this end, a suitable starting strain, such as for example ATCC 14752 or ATCC 13032 is subjected to a mutagenesis process.
Classical mutagenesis processes are treatment with chemicals such as for example N-methyl-N-nitro-N-nitrosoguanidine or UV irradiation. Methods of this type for initiating mutation are generally known and may be found inter alia in Miller. (A Short Course in Bacterial Genetics, A Laboratory Manual and Handbook for Escherichia coli and Related Bacteria (Cold Spring Harbor Laboratory Press, 1992)) or in the manual xe2x80x9cManual of Methods for General Bacteriologyxe2x80x9d of the American Society for Bacteriology (Washington D.C., USA, 1981).
Another mutagenesis method is the transposon mutagenesis method which exploits the characteristic of a transposon to xe2x80x9cjumpxe2x80x9d into DNA sequences, so disrupting or suppressing the function of the gene concerned. Transposons of coryneform bacteria are known in specialist circles. The erythromycin resistance tfansposon Tn5432 (Tauch et al., Plasmid (1995) 33: 168-179) and the chloramphenicol resistance transposon Tn5546 have accordingly been isolated from Corynebacterium xerosis strain M82B. Tauch et al. (Plasmid (1995) 34: 119-131 and Plasmid (1998) 40: 126-139) demonstrated that mutagenesis is possible with these transposons.
Another transposon is transposon Tn5531, which is described in Ankri et al. (Journal of Bacteriology (1996) 178: 4412-4419) and was used by way of example in the course of the present invention. Transposon Tn553l contains the aph3 kanamycin resistance gene and may be administered in form of the plasmid vector pCGL0040, which is shown in FIG. 1. The nucleotide sequence of transposon Tn5531 is freely available under the accession number U53587 from the National Center for Biotechnology Itformation (NCBI, Bethesda, Md., USA).
Once mutagenesis, preferably transposon mutagenesis, has been performed, a mutant defective with regard to brnF or brnE is sought. A mutant defective with regard to brnF or brnE is recognised by the fact that it exhibits good growth on minimal agar, but poor growth on minimal agar which has been supplemented with oligopeptides containing branched-chain amino acids, such as for example the dipeptide isoleucyl-isoleucine.
One example of such a mutant is strain ATCC14752brnE::Tn5531.
A strain produced in the stated manner may be used for cloning and sequencing the brnF and/or brnE gene.
To this end, a gene library of the bacterium under consideration may be constructed. The construction of gene libraries is described in generally known textbooks and manuals. Examples which may be mentioned are the textbook by Winnacker, Gene und Klone, Eine Einfxc3xchrung in die Gentechnologie (Verlag Chemie, Weinheim, Germany, 1990) or the manual by Sambrook et al., Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1989). One very well known gene library is that of E. coli K-12 strain W3110, which was constructed by Kohara et al. (Cell 50, 495-508 (1987)) in xcex-vectors. Bathe et al. (Molecular and General Genetics, 252:255-265, 1996) describe a gene library of C. glutamicum ATCC13032, which was constructed using the cosmid vector SuperCos I (Wahl et al., 1987, Proceedings of the National Academy of Sciences USA, 84:2160.2164) in E. coli K-12 strain NM554 (Raleigh et al., 1988, Nucleic Acids Research 16:1563-1575). Vectors suitable for the present invention are those which replicate in coryneform bacteria, preferably Corynebacterium glutamicum. Such vectors are known from the prior art; one example which may be mentioned is the plasmid vector pZ1, which is described in Menkel et al. (Applied and Environmental Microbiology (1989) 64: 549-554). The gene library obtained in the stated manner is then transferred by transformation or electroporation into the indicator strain which is defective with regard to brnF or brnE and those transformants are sought which are capable of growing on minimal agar in the presence of oligopeptides containing branched-chain amino acids. The cloned DNA fragment may then be subjected to sequence analysis.
When a mutant produced by Tn5531 mutagenesis of a coryneform bacterium, such as for example strain ATCC 14752brnE::Tn5531, is used, the brnE::Tn5531 allele may be directly cloned and isolated by exploiting the kanamycin resistance gene aph3 contained therein. Known cloning vectors, such as for example pUC18 (Norrander et al., Gene (1983) 26: 101-106 and Yanisch-Perron et al., Gene (1985) 33: 103-119) are used for this purpose. Suitable cloning hosts are in particular those strains of E. coli with restriction and recombination defects. One example of such a strain is the strain DH5xcex1mcr, which has been described by Grant et al. (Proceedings of the National Academy of Sciences USA, 87 (1990) 4645-4649). Transformant selection proceeds in the presence of kanamycin. The plasmid DNA of the resultant transformants is then sequenced. The dideoxy chain termination method of Sanger et al. (Proceedings of the National Academy of Sciences of the United States of America USA (1977) 74: 5463-5467) may be used for this purpose. Using this method, the genes located upstream and downstream from the Tn5531 insertion site are obtained. The nucleotide sequences obtained are then analysed and assembled using commercially available sequence analysis software, such as for example the Lasergene package (Biocomputing Software for Windows, DNASTAR, Madison, USA) or the HUSAR package (release 4.0, EMBL, Heidelberg, Germany).
This is the method which was used to obtain the novel DNA sequences of C. glutamicum which code for the export of branched-chain amino acids and are provided by the present invention as SEQ ID no. 1. SEQ ID no. 2 and SEQ ID no. 4 show the coding regions of the genes brnF and brnE. SEQ ID no. 3 and SEQ ID no. 5 show the amino acid sequences of the gene products obtained respectively from SEQ ID no. 1 or from SEQ ID no. 2 and SEQ ID no. 4.
Coding DNA sequences arising from the degeneracy of the genetic code are also provided by the present invention. DNA sequences which hybridise with SEQ ID no. 1 or parts of SEQ ID no. 1 are similarly provided by the invention. Conservative substitutions of amino,acids in proteins, for example the substitution of glycine for alanine or of aspartic acid for glutamic acid, are known in specialist circles as xe2x80x9csense mutationsxe2x80x9d, which result in no fundamental change in activity of the protein, i.e. they are functionally neutral. It is furthermore known that changes to the N and/or C terminus of a protein do not substantially impair or may even stabilise the function thereof. The person skilled in the art will find information in this connection inter alia in Ben-Bassat et al. (Journal of Bacteriology 169:751-757 (1987)), in O""Regan et al. (Gene 77:237-251 (1989)), in Sahin-Toth et al. (Protein Sciences 3:240-247 (1994)), in Hochuli et al. (Bio/Technology 6:1321-1325 (1988)) and in known textbooks of genetics and molecular biology. Amino acid sequences arising in a corresponding manner from SEQ ID no. 2 or SEQ ID no. 4 are also provided by the present invention.
Using the nucleotide sequence shown in SEQ ID no. 1, it is possible to synthesise suitable primers and these may then be used with the assistance of the polymerase chain reaction (PCR) to amplify the brnF and brnE genes of various coryneform bacteria and strains. The person skilled in the art will find instructions in connection inter alia in the textbook by Gait, Oligonucleotide synthesis: a practical approach (IRL Press, Oxford, UK, 1984) and in Newton and Graham, PCR (Spektrum Akademischer Verlag, Heidelberg, Germany, 1994). Alternatively, the nucleotide sequence shown in SEQ ID no. 1 or parts thereof may be used as a probe to search for brnF and/or brnE genes in gene libraries, in particular of coryneform bacteria. The person skilled in the art will find instructions in this connection inter alia in the manual xe2x80x9cThe DIG System Users Guide for Filter Hybridizationxe2x80x9d from Boehringer Mannheim GmbH (Mannheim, Germany, 1991) and in Liebl et al. (International Journal of Systematic Bacteriology (1991) 41: 255-260). DNA fragments containing brnE and brnF genes amplified in this manner are then cloned and sequenced.
The DNA sequence of the genes brnF and brnE of strain ATCC 13032 shown in SEQ ID no. 6 was obtained in this manner and is also provided by the present invention.
The inventors discovered that coryneform bacteria produce branched-chain amino acids in an improved manner once the brnF and/or brnE export gene has been overexpressed.
Overexpression may be achieved by increasing the copy number of the corresponding genes or by mutating the promoter and regulation region or the ribosome-binding site located upstream from the structural gene. Expression cassettes incorporated upstream from the structural gene act in the same manner. It is additionally possible to increase expression during the fermentative production of branched-chain amino acids by inducible promoters. Expression is also improved by measures to extend the lifetime of the mRNA. Enzyme activity is moreover amplified by preventing degradation of the enzyme protein. The genes or gene constructs may either be present in plasmids in a variable copy number or be integrated in the chromosome and amplified. Alternatively, overexpression of the genes concerned may also be achieved by modifying the composition of the nutrient media and culture conditions.
The person skilled in the art will find guidance in this connection inter alia in Martin et al. (Bio/Technology 5, 137-146 (1987)), in Guerrero et al. (Gene 138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430 (1988)), in Eikmanns et al. (Gene 102, 93-98 (1991)), in European patent EP-B 0 472 869, in U.S. Pat. No. 4,601,893, in Schwarzer and Pxc3xchler (Bio/Technology 9, 84-87 (1991)), in Reinscheid et al. (Applied and Environmental Microbiology 60, 126-132 (1994)), in LaBarre et al. (Journal of Bacteriology 175, 1001-1007 (1993)), in patent application WO 96/15246, in Malumbres et al. (Gene 134, 15-24 (1993)), in Japanese published patent application JP-A-10-229891, in Jensen and Hammer (Biotechnology and Bioengineering 58, 191-195 (1998)), in Makrides (Microbiological Reviews 60:512-538 (1996)) and in known textbooks of genetics and molecular biology.
By way of example, the genes brnF and brnE according to the invention were overexpressed with the assistance of plasmids. Suitable plasmids are those which are replicated in coryneform bacteria. Numerous known plasmid vectors, such as for example pZ1 (Menkel et al., Applied and Environmental Microbiology (1989) 64: 549-554), pEKEx1 (Eikmanns et al., Gene 102:93-98 (1991)) or pHS2-1 (Sonnen et al., Gene 107:69-74 (1991)) are based on the cryptic plasmids pHM1519, pBL1 or pGA1. Other plasmid vectors, such as for example those based on pCG4. (U.S. Pat. No. 4,489,160), or pNG2 (Serwold-Davis et. al., FEMS Microbiology Letters 66, 119-124 (1990)), or pAG1 (U.S. Pat. No. 5,158,891) may be used in the same manner.
It may additionally be advantageous for the production of branched-chain amino acids, in addition to novel brnF and brnE genes, to overexpress one or more genes which code for further enzymes of the known biosynthetic pathway of branched-chain amino acidsor enzymes of anaplerotic metabolism, or enzymes of the citric acid cycle.
Thus, for example, for theproduction of L-isoleucine
the hom gene (Peoples et al., Molecular Microbiology 2, 63-72 (1988)) which codes for homoserine dehydrogenase or the homdr allele (Archer et al., Gene 107, 53-59 (1991)) which codes for a xe2x80x9cfeed back resistantxe2x80x9d homoserine dehydrogenase may simultaneously be overexpressed or
the ilvA gene (Mxc3x6ckel et al., Journal of Bacteriology (1992) 8065-8072)) which codes for threonine dehydratase or the ilvA(Fbr) allele (Mxc3x6ckel et al., (1994) Molecular Microbiology 13: 833-842) which codes for a xe2x80x9cfeed back resistantxe2x80x9d threonine dehydratase may simultaneously be overexpressed or
the genes ilvBN (Keilhauer et al., (1993) Journal of Bacteriology 175: 5595-5603) which code for acetohydroxy acid synthase may simultaneously be overexpressed or
the ilvD gene (Sahm und Eggeling (1999) Applied and Environmental Microbiology 65: 1973-1979) which codes for dihydroxy acid dehydratase may simultaneously be overexpressed or
the pyc gene (DE-A-19 831 609) which codes for pyruvate carboxylase may simultaneously be overexpressed or
the mqo gene (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)) which codes for malate:quinone oxidoreductase may simultaneously be overexpressed.
Thus, for example, for the production of L-leucine,
the leuA gene (Pxc3xa1tek et al., Applied Environmental Microbiology 60 (1994) 133-140) which codes for isopropyl malate synthase or an allele which codes for a xe2x80x9cfeed back resistantxe2x80x9d isopropyl malate synthase may simultaneously be overexpressed or
the leuC and leuD genes (Pxc3xa1tek et al., Applied Environmental Microbiology 60 (1994) 133-140) which code for isopropyl malate dehydratase may simultaneously be overexpressed or
the leuB gene (Pxc3xa1tek et al., Applied Environmental Microbiology 60 (1994) 133-140) which codes for isopropyl malate dehydrogenase may simultaneously be overexpressed or
the genes ilvBN (Keilhauer et al., (1993) Journal of Bacteriology 175: 5595-5603) which code for acetohydroxy acid synthase may simultaneously be overexpressed or
the ilvD gene (Sahm und Eggeling (1999) Applied and Environmental Microbiology 65: 1973-1979) which codes for dihydroxy acid dehydratase may simultaneously be overexpressed or
the mqo gene (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)) which codes for malate:quinone oxidoreductase may simultaneously be overexpressed.
Thus, for example, for the production of L-valine
the genes ilvBN (Keilhauer et al., (1993) Journal of Bacteriology 175: 5595-5603) which code for acetohydroxy acid synthase may simultaneously be overexpressed or
the ilvD gene (Sahm und Eggeling (1999) Applied and Environmental Microbiology 65: 1973-1979) which codes for dihydroxy acid dehydratase may simultaneously be overexpressed or
the mqo gene (Molenaar et al., European Journal of Biochemistry 254, 395-403 (1998)) which codes for malate:quinone oxidoreductase may simultaneously be overexpressed.
It may furthermore be advantageous for the production of branched-chain amino acids, in addition to overexpressing the brnE and/or brnF gene, to suppress unwanted secondary reactions (Nakayama: xe2x80x9cBreeding of Amino Acid. Producing Microorganismsxe2x80x9d, in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vahek (eds.), Academic Press, London, UK, 1982).
For the purposes of branched-chain amino acid production, the microorganisms according to the invention may be cultured continuously or discontinuously using the batch process or the fed batch process or repeated fed batch process. A summary of known culture methods is given in the textbook by Chmiel (Bioprozesstechnik 1. Einfxc3xcihrung in die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).
The culture medium to be used must adequately satisfy the requirements of the particular strains. Culture media for various microorganisms are described in xe2x80x9cManual of Methods for General Bacteriologyxe2x80x9d from American Society for Bacteriology (Washington D.C., USA, 1981). Carbon sources which may be used include sugars and carbohydrates, such as for example glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as for example soya oil, sunflower oil, peanut oil and coconut oil, fatty acids, such as for example palmitic acid, stearic acid and linoleic acid, alcohols, such as for example glycerol and ethanol, and organic acids, such as for example acetic acid. These substances may be used individually or as a mixture. Nitrogen sources which may be used comprise organic compounds containing nitrogen, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya flour and urea or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. The nitrogen sources may be used individually or as a mixture. Phosphorus sources which may be used are phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding salts containing sodium. The culture medium must furthermore contain metal salts, such as for example magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth-promoting substances such as amino acids and vitamins may also be used in addition to the above-stated substances. Suitable precursors may furthermore be added to the culture medium. The stated feed substances may be added to the culture as a single batch or be fed appropriately during cultivation.
Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water, or acidic compounds, such as phosphoric acid or sulfuric acid, are used appropriately to control the pH of the culture. Antifoaming agents, such as for example fatty acid polyglycol esters, may be used to control foaming. Suitable selectively acting substances, such as for example antibiotics, may be added to the medium in order to maintain plasmid stability. Oxygen or gas mixtures containing oxygen, such as for example air, are introduced into the culture in order to maintain aerobic conditions. The temperature of the culture is normally from 20xc2x0 C. to 45xc2x0 C. and preferably from 25xc2x0 C. to 40xc2x0 C. The culture is continued until the maximum quantity of branched-chain amino acids has formed. This objective is normally achieved within 10 hours to 160 hours.
The branched-chain amino acids may be analysed by anion exchange chromatography with subsequent ninhydrin derivatisation, as described in Spackman et al. (Analytical Chemistry, 30, (1958), 1190) or by reversed phase HPLC, as described in Lindroth et al. (Analytical Chemistry (1979) 51: 1167-1174).
The following microorganism has been deposited with Deutschen Sammlung fxc3xcr Mikrorganismen und Zellkulturen (DSMZ, Braunschweig, Germany) in accordance with the Budapest Treaty:
Escherichia coli strain GM2929pCGL0040 as DSM 12839